Microelectrode arrangement

ABSTRACT

The invention concerns a microelectrode arrangement for leaking, with local resolution, electrical cell potentials, or for electrical stimulation of networks of biological cells such as for example cell cultures, tissue slices &#34;in vitro&#34; or biological tissue &#34;in vivo&#34;. In order to attain high local resolution and high temporal resolution, the invention suggests that, as microelectrodes (M 1  to M n ), in each instance a contacting electrode (K 1  to K n ) be placed above a pad electrode (A 1  to A n ) onto a substrate (S); with light-sensitive elements, preferably in the shape of a continuous layer (P) arranged between the said contacting electrodes and the pad electrodes. By illuminating the light-sensitive layer (P) in the region of individual microelectrodes (M 1  to M n ), these microelectrodes are selected. Selection is preferably by the transmitted-light process through the substrate (S). In this case, substrate (S) and pad electrodes (A 1  to A n ) must be translucent. For selection by means of impinging light, the contacting electrodes (K 1  to K n ) are constructed in a translucent way (FIG. 1b).

The invention concerns a microelectrode arrangement forlocally-resolved, in particular extracellular, leakage measurement ofelectrical cell potentials or for electrical stimulation of networks ofbiological cells.

Biological cells or networks of biological cells such as for examplecell cultures, tissue slices "in vitro" or biological tissue "in vivo"in electrophysiology are usually contacted by glass microelectrodes withelectrolyte filling, or by metal microelectrodes. The electrodes areinserted into a cell by means of a so-called micromanipulator(intracellular process), brought into close contact with a cell membrane(patch clamp process), or brought into the vicinity of the cell membrane(extracellular process) so that the microelectrodes are connected in anelectrically-conductive way to the biological cells of the network byway of an electrolyte solution. The disadvantage of these contactingprocesses is that only one, or with great expense, only a few cells canbe contacted simultaneously and as a result, no network characteristicscan be examined.

For this reason attempts have been made in recent times to contact anetwork of biological cells at many places concurrently, by means ofmicrostructured microelectrodes deposited onto a substrate (carrier) bymethods known from microelectronics, to leak electrical cell potentialsin an extracellular way or to be able to electrically stimulate thecells. In this, the microelectrodes should be arranged in the highestpossible density in order to achieve high local resolution. Furthermore,the electrical potentials of the cells should be leaked as far aspossible simultaneously, i.e. in a parallel way, or electricalpotentials for stimulating the network should be able to be applied tothese cells simultaneously, in order to achieve a high temporalresolution.

There is however the problem that electrical leads from the individualmicroelectrodes have to be conducted in an insulated way right up to anelectronic device for measuring or stimulation, or similar. Themultitude of parallel leads insulated from each other limits the localresolution of the microelectrode arrangement.

Another option is, for each microelectrode to accommodate an integratedelectronic switch on the substrate and to connect (select) themicroelectrodes to the measuring or stimulation electronics by multiplexoperation individually or in groups, in time sequence. This necessitatesvery great expense in integrated circuit engineering (VLSI technology)and thus considerably increases the cost of the microelectrodearrangement. In addition, the local resolution remains limited, due tothe electronic switches which need to be accommodated on the substrate.Furthermore, the microelectrodes can no longer be selected concurrently,but only individually or in groups in sequence; the temporal resolutionof the leakage or stimulation is reduced. Interference voltages are afurther disadvantage; when switching, they can be transmitted by theelectronic switches to the microelectrodes and to their connecting leadsand can be superimposed on the measuring signal. Such interferencevoltages negatively influence the measurement result and thesignal-to-noise ratio. Interference voltages can exceed the measuringsignal many times, therefore their decay must be awaited afterswitching, before any measurements or stimulation can take place at all.This further reduces the temporal resolution of the microelectrodearrangement.

The number of microelectrodes of known microelectrode arrangements istherefore limited (less than 100 microelectrodes).

It is thus the object of the invention to provide a microelectrodearrangement of the type mentioned in the introduction, with a very largenumber of microelectrodes which as a result of small dimensions of themicroelectrodes and small spacing from each other allows a high localresolution and in addition a high temporal resolution.

This task is solved by the characteristics of claims 1 and 9. Eachmicroelectrode of the microelectrode arrangement according to theinvention comprises a contacting electrode, a connection for anelectronic device for measuring or stimulation, or similar, belowreferred to as a "pad electrode", as well as a light-sensitive element.

By way of an electrolyte solution, the contacting electrode can bebrought into electrically conducting contact with a biological cell of anetwork. This preferably takes place in that the microelectrodearrangement is brought to a network of biological cells and thus themicroelectrodes are brought into close proximity of cell membranes, i.e.in an extracellular way. In this, an electrical transition resistance(impedance) exists between the cells and the microelectrodes.

The light-sensitive element which in darkness has a very high electricalresistance which is reduced when it is exposed to light (or vice versa),is arranged between the contacting electrode and the pad electrode; thesaid light-sensitive element serves as a switch insulating thecontacting electrode from the pad electrode or connecting it with thepad electrode as an ohmic resistance. This switch is activated bydirecting light onto it, i.e. onto the light-sensitive element. Thuseach microelectrode is individually selectable by light; themicroelectrodes of the microelectrode arrangement according to theinvention can be addressed by light.

The invention provides the advantage that its microelectrodes are ofvery small dimensions and can be arranged very closely beside eachother, thus a high local resolution can be achieved. The inventionprovides a further advantage in that the microelectrodes can be selectedsimultaneously, i.e. in a parallel way, individually or in groups, thusallowing high temporal resolution. A further advantage is provided inthat due to selection by light, no interference voltages occur which aresuperimposed onto the measurement signal, and the decay of which wouldhave to be awaited prior to taking a measurement or undertaking astimulation.

The contacting electrodes, the light-sensitive element and theconnecting electrodes can be arranged in two or three planes above eachother or else in one plane beside each other, on a substrate. Thearrangement in three planes above each other results in the most closelyspaced arrangement of the microelectrodes, and thus provides the highestlocal resolution.

The light-sensitive element which preferably when it is not impinged bylight, i.e. when it is dark, provides electrical insulation, can serveto insulate the contacting electrodes and the pad electrodes of thevarious microelectrodes from each other. In this case, thelight-sensitive element is designed as a continuous layer common to allmicroelectrodes or to groups of microelectrodes onto which light isdirected which is locally limited to the microelectrodes to be selected.In this case, either the contacting electrode or the pad electrode andthe substrate onto which the microelectrodes are deposited, must betranslucent for selection by light.

If the light-sensitive elements are arranged beside the contactingelectrodes or beside the connecting electrodes, then the contactingelectrodes and the pad electrodes can be made light impermeable, fromthe same material and deposited onto the substrate in one work process.

The pad electrodes of all microelectrodes or of groups of themicroelectrodes can be combined in a common pad electrode. In this waythe required number of connecting leads is reduced, however themicroelectrodes can no longer be selected in parallel but only in seriesor parallel in groups.

In order to select the microelectrodes, one embodiment of the inventionprovides for fibreoptics which preferably comprise the same number ofoptic fibres as there are microelectrodes in the arrangement, so thatone optic fibre leads to each microelectrode. In this, the front ends ofthe optic fibres from which light emanates can serve as the substratefor the microelectrodes.

In a further development of the invention, the fibreoptics comprise alight source for each optic fibre. Preferably the light sources arelight-emitting diodes which are combined to form a matrix.

The microelectrode arrangement according to the invention can beimplanted for leaking impulses or for electrical stimulation of nervecells in plants or living things. For example, the microelectrodearrangement according to the invention can be used as a retina implant.

Focused light, for example a laser beam, is used to select certainmicroelectrodes of the arrangement according to the invention. Patternsof light spots, light beams or similar can be projected onto thearrangement in order to simultaneously select particularmicroelectrodes.

Below, the invention is explained in greater detail by means of anexemplary embodiment, as follows:

FIG. 1 shows a section through a microelectrode arrangement according tothe invention with microelectrodes to be selected in series (FIG. 1a) orin parallel (FIG. 1b);

FIG. 2 shows a diagrammatic representation of a microelectrodearrangement according to the invention (FIG. 2a in series, FIG. 2b inparallel);

FIG. 3 shows a top view of a microelectrode arrangement according to theinvention, with microelectrodes to be switched in a column-parallel wayand to be selected in a line-parallel way; and

FIG. 4 shows a section along line IV--IV in FIG. 3.

The microelectrode arrangement 10, according to the invention, shown inFIGS. 1a and b has been deposited onto a substrate S. Preferably, thesubstrate S comprises a translucent material, such as for example glassor plastic. It can however also comprise a non-translucent material suchas for example ceramics or silicon with oxide layer insulation, as isknown for example from microelectronics.

The microelectrodes M₁ to M_(n) comprise pad electrodes A, A₁ to A_(n),light-sensitive elements P and contacting electrodes K₁ -K_(n), whichhave been deposited as thin-film elements onto the substrate S on top ofeach other in three planes in the order mentioned. With serial selectionof the microelectrodes M₁ to M_(n), a single, continuous pad electrodeA, shared by all microelectrodes M₁ to M_(n), can be deposited onto thesubstrate S (FIG. 1a). With parallel selection, each microelectrode M₁to M_(n) comprises a pad electrode A₁ to A_(n), which are all separatedfrom each other by an insulating layer I. The insulating layer I hasbeen deposited in one layer with the pad electrodes A₁ to A_(n), ontothe substrate S.

The light-sensitive elements have been deposited as a continuous layer Pin common for all microelectrodes M₁ to M_(n), onto the pad electrodesA, A₁ to A_(n) and if applicable to the insulating layer I. Contactingelectrodes K₁ to K_(n), which during parallel selection are locatedabove the pad electrodes A₁ to A_(n), have been deposited onto the layerP constituting the light-sensitive elements. The contacting electrodesK₁ to K_(n) which have been deposited onto the light-sensitive layer Pin a plane with the contacting electrodes K₁ to K_(n) are also separatedfrom each other by an insulating layer. The contacting electrodes K₁ toK_(n) protrude slightly above their insulating layer I.

The contacting electrodes K₁ to K_(n), designed as thin-film elements,the light-sensitive elements P and the pad electrodes A, A₁ to A_(n) aredeposited onto the substrate S by vapour-depositing, sputtering or PECVD(plasma-enhanced chemical vapour deposition) and microstructured byphotolithographic methods.

The pad electrodes A, A₁ to A_(n) comprise a material with goodelectrical conductivity, preferably translucent, such as for exampleindium tin oxide (ITO) or zinc oxide (ZnO).

The light-sensitive elements designed as a continuous layer P can bedesigned as thin-film photo resistors, photo diodes with PN junctions orPIN junctions or as photo transistors. These may be made in thin-filmtechnology from materials such as for example amorphous silicon (Si),cadmium sulfide (CdS) or cadmium selenide (CdSe).

The contacting electrodes K₁ to K_(n) preferably comprise abiocompatible, conductive material such as for example gold (Au),platinum (Pt), titanium (Ti), iridium (Ir) and are insulated from eachother by the biocompatible insulating layer I, for example made fromsilicon oxide, silicon nitride or polyamide. The contacting electrodescan also be made from translucent material as used for the padelectrodes A, A₁ to A_(n). Equally, the pad electrodes A, A₁ to A_(n)may be made light-impermeable from the same material as the contactingelectrodes K₁ to K_(n).

In the embodiment of the invention shown in FIG. 1a, a common lead forall microelectrodes M₁ to M_(n) has been deposited for connection to anelectronic device for measuring or stimulation, or similar, at thecommon continuous pad electrode A, preferably at its marginal area (notshown). In the case of the embodiment of the invention shown in FIG. 1b,the said pad electrodes A₁ to A_(n) insulated from each other, each havetheir own connecting lead (not shown).

The diagrammatic representation in FIGS. 2a and b shows the applicationof the microelectrode arrangements 10 from FIGS. 1a and b for leakingelectrical cell potentials or for electrical stimulation of networks ofbiological cells Ze. The biological cells Ze are contained in acylindrical culture vessel Ge in a physiological electrolyte E. Thesubstrate S with the microelectrode arrangement M₁ to M_(n) from FIGS.1a and b constitutes the bottom of the culture vessel Ge. In this, thecontacting electrodes K₁ to K_(n) not shown in detail in FIGS. 2a and b,are located in close proximity to cell membranes of the cells Ze and arethus each connected to a cell Ze (extracellular connection), in anelectrically conducting way via the electrolyte, with an electricalresistance (impedance) present between the cell Ze and the contactingelectrode K₁ to K_(n) of the respective microelectrodes M₁ to M_(n).

A reference electrode Re made of metal is immersed into thephysiological electrolyte E so that by means of the microelectrodes M₁to M_(n), an electrical potential can be measured at each desiredposition in the network of biological cells Ze, or by means of themicroelectrodes M₁ to M_(n), the network of biological cells Ze can beelectrically stimulated at all desired locations.

The light-sensitive elements P₁ to P_(n) and pad electrodes A, A₁ andA_(n), deposited onto the substrate S, are represented in FIGS. 2a and bwith their connecting leads Z, Z₁ to Z_(n) in the form of an electricalcircuit diagram.

FIGS. 3 and 4 show a microelectrode arrangement 10 according to theinvention with microelectrodes M₁ to M_(n) switched in a column-parallelway, where the section according to FIG. 4 corresponds to FIGS. 1a andb. The structure and arrangement of the contacting electrodes K₁ toK_(n) which are insulated from each other by an insulating layer I, andthe light-sensitive thin film P corresponds to the above mentionedarrangement, as shown in FIGS. 1a and b. FIG. 3 shows the matrix-shapedarrangement of the micro electrodes M₁ to M_(n). Pad electrodes A₁ toA_(n) are shaped as parallel conducting tracks, continuous in columndirection, which on one margin of the substrate S enlarge to becomecontacting surfaces Z₁ to Z_(s). On the contacting surfaces Z₁ to Z_(s),connecting cables for connecting the microelectrode arrangement 10 tomeasuring or stimulation electronics are soldered, welded or fixed insome other known way so as to provide electrical conduction. In theembodiment according to FIGS. 3 and 4, the microelectrodes M₁ to M_(n)are brought together into groups comprising a column each. Instead ofcolumns, it is possible, for example, to bring together circles or othergroups of microelectrodes M₁ to M_(n).

The pad electrodes A₁ to A_(s) are separated from each other by aninsulating layer I. The same materials as mentioned in FIGS. 1a and bcan be used as materials for the contacting electrodes K₁ to K_(n), thelight-sensitive layer P, the pad electrodes A₁ to A_(n), the insulatinglayers I and the substrate.

When the microelectrodes M₁ to M_(n) are switched in a column-parallelway, only one microelectrode M₁ to M_(n) of each column can be selectedat a given time, i.e. leakage or stimulation can take place. Selectioncan take place line-by-line or by some other pattern.

Selection of the microelectrode arrangement 10 according to theinvention, which selection is shown below in FIG. 3, is by a focussed orshaped light beam or a projected light image which for example isgenerated by using a laser or provided to the microelectrodes M₁ toM_(n) by means of glass fibres. For selection, the light-sensitive layerP in the region of one or several microelectrodes M₁ to M_(n) to beselected, is illuminated. The illuminated region forms thelight-sensitive element of the respective microelectrode M₁ to M_(n).The illuminated region of the light-sensitive layer P becomeselectrically conductive, so that the contacting electrodes K₁ to K_(n)of the selected microelectrodes M₁ to M_(n) are connected in anelectrically conductive way with the respective pad electrode A₁ toA_(s) and that the electrical potential of a biological cell in closeproximity to the respective microelectrode M₁ to M_(n) (FIGS. 2a and b)is leaked, i.e. measured, or that the biological cell can beelectrically stimulated.

Selection is either by means of impinging light, i.e. through thenetwork of biological cells, from the direction of the side of thecontacting electrodes K₁ to K_(n). In this case, the contactingelectrodes K₁ to K_(n) must be translucent or be arranged laterallybeside the light-sensitive layer P forming the light-sensitive elementwhich separates the said contacting electrodes K₁ to K_(n) from theirpad electrode A₁ to A_(s). Equally, selection can be by transmittedlight from the direction of the side of the substrate S. In this casethe substrate S and the pad electrodes A₁ to A_(s) must be translucentor arranged beside the light-sensitive layer P forming thelight-sensitive element separating them from the contacting electrodesK₁ to K_(n). In the region not illuminated, the thin film P providesinsulation. Thus, by locally limited illumination in the region of amicroelectrode M₁ to M_(n) in the illuminated region it constitutes thelight-sensitive element of this microelectrode M₁ to M_(n).

When using amorphous silicon, resistance ratios of up to five powers often between illuminated (bright) and non-illuminated (dark) areattained. With a microelectrode M₁ to M_(n) with a surface of 10 μm by10 μm and a thickness of 0.1 μm, at a dark conductivity of Sigma=10⁻⁹(ohms×cm)⁻¹, a dark resistance of 10¹⁰ 1/2 and with exposure to light, alight resistance of 10⁵ 1/2. At the surface mentioned of 10 μm×10 μm, acontacting electrode K₁ to K_(n), has a resistance through theelectrolyte E to the biological cell Ze of also approximately 10⁵ 1/2;the said resistance being determined by the Helmholtz double layer atthe boundary surface metal/electrolyte. With light incidence onto thelight-sensitive element P, there is a total transition resistance fromthe biological cell Ze to the pad electrode A₁ to A_(s) of approx. 2×10⁵1/2. By contrast, the total transition resistance with a darklight-sensitive element P is approximately 10¹⁰ 1/2. There is a goodcontact/separation ratio by the light/dark sampling of themicroelectrodes M₁ to M_(n) in respect of their selection.

Since the spacing between the microelectrodes M₁ to M_(n) is large whencompared to the layer thickness of the light-sensitive layer P,insulation of the light-sensitive elements constituted by the saidlight-sensitive layer P from each other is not necessary and it can beconstructed in a continuous layer P as described and illustrated. In theembodiment of the invention shown in FIG. 3, selection of themicroelectrodes M₁ to M_(n) is by means of a light beam L, aligned inthe direction of the line, i.e. transverse to the pad electrodes A₁ toA_(s) ; the said light beam L illuminates the light-sensitive elementsof microelectrodes M₁ to M_(n) which are arranged in a line. Thus themicroelectrodes M₁ to M_(n) of a line are selected simultaneously andthe electrical cell potentials of the biological cells Ze contacted bythese are leaked by way of pad electrodes A₁ to A_(s), or thesebiological cells Ze are stimulated electrically. The light beam L ismobile in the direction of the column (double arrow in FIG. 3).Selection can of course also take place in various lines, that is not bymeans of a light beam but by means of individual light spots directedonto individual microelectrodes M₁ to M_(n). In this, from each column,only one microelectrode M₁ to M_(n) can be selected at a given point intime. If the spacing of the microelectrodes M₁ to M_(n) is notsufficient, so that the signals of adjacent microelectrodes M₁ to M_(n)in the region illuminated by the light beam L and thus conductive regionof the light-sensitive layer P influence each other, then no continuouslight beam L can be used for selecting the microelectrodes M₁ to M_(n).Instead, there must always remain a dark region between themicroelectrodes M₁ to M_(n), or else an additional insulating layer mustbe provided in the light-sensitive layer P (not shown) between the padelectrodes A₁ to A_(s).

In the case of a microelectrode surface of 10 μm×10 μm and at 20 μmelectrode spacing, there are for example 60 columns, each with 60microelectrodes, i.e. a total of 3600 microelectrodes M₁ to M_(n) on asubstrate field with a surface of 1.8 mm×1.8 mm.

In the case of a microelectrode arrangement, if applicable, selection ofthe light-sensitive elements can also be by means of a light-emittingdiode matrix as a substrate or by a projected light image.

I claim:
 1. A microelectrode arrangement for leaking, with localresolution, electrical cell potentials, or for electrical stimulation ofnetworks of biological cells, with a multitude of microelectrodes,wherein each microelectrode comprises:a contacting electrode having afirst and a second surface and being adapted for electrically contactingthe network of biological cells at its first surface; a pad electrodewhich is connectable in an electrically conductive way to a measuringdevice; a thin-film photoresistor having a first side and a second sideand being arranged between the contacting electrode and the padelectrode, said thin-film photoresistor contacting said second surfaceof said contacting electrode at its first side and contacting said padelectrode at its second side.
 2. A microelectrode arrangement accordingto claim 1, including fiberoptics having at least one optic fiberarranged in front of one of said microelectrodes for emitting light ontosaid thin-film resistor for optically activating said one of saidmicroelectrodes.
 3. A microelectrode arrangement according to claim 2,wherein said fiberoptics comprise a plurality of optical fibers, eachone of said microelectrodes having one of said optical fibers associatedtherewith for optically activating said one of said microelectrodes. 4.A microelectrode arrangement according to claim 3, wherein each opticalfiber is associated with a light source for emitting light into saidoptical fiber.
 5. A microelectrode arrangement according to claim 1,wherein a focused light beam is directed in a locally limited way onto alight-sensitive element (P) of at least one microelectrode (M_(n) toM_(n)).
 6. A microelectrode arrangement according to claim 1, whereinselection takes place by at least one of a light-emitting diode matrixas a substrate, and by a projected light image.
 7. A microelectrodearrangement according to claim 1, used as an implant.
 8. Amicroelectrode arrangement for leaking, with local resolution,electrical cell potentials, or for electrical stimulation of networks ofbiological cells, said microelectrode arrangement comprising a multitudeof microelectrodes, wherein:each microelectrode comprises a contactingelectrode having a first and a second surface and being adapted forelectrically contacting the network of biological cells at its firstsurface; a common pad electrode is provided for electrical connection toa measuring device; a thin-film photoresistor having a first side and asecond side is arranged between the contacting electrodes and the commonpad electrode, said thin-film photoresistor contacting said secondsurfaces of said multitude of contacting electrodes at its second side.9. A microelectrode arrangement according to claim 8, includingfiberoptics having at least one optic fiber arranged in front of one ofsaid microelectrodes for emitting light onto said thin-film resistor foroptically activating said one of said microelectrodes.
 10. Amicroelectrode arrangement according to claim 9, wherein saidfiberoptics comprise a plurality of optical fibers, each one of saidmicroelectrodes having one of said optical fibers associated therewithfor optically activating said one of said microelectrodes.
 11. Amicroelectrode arrangement according to claim 9, wherein each opticalfiber is associated with a light source for emitting light into saidoptical fiber.
 12. A microelectrode arrangement for leaking, with localresolution, electrical cell potentials, or for electrical stimulation ofnetworks of biological cells, said microelectrode arrangement comprisinga multitude of microelectrodes arranged side-by-side, a first pluralityof which forming a first group, a second plurality of which forming asecond group containing at least one microelectrode, wherein:eachmicroelectrode comprises a contacting electrode having a first and asecond surface and being adapted for electrically contacting the networkof biological cells at its first surface; a common pad electrode adaptedfor electrical connection to an external device is provided; at leastone pad electrode adapted for electrical connection to an externaldevice is arranged side-by-side to said common pad electrode; athin-film photoresistor having a first side and a second side isprovided, said thin-film photoresistor having a first section and atleast one second section, said first section being arranged between saidfirst group of contacting electrodes and said common pad electrode, saidfirst section contacting said second surfaces of said first group ofcontacting electrodes at a first side thereof and contacting said commonpad electrode at a second side thereof; each of said second plurality ofmicroelectrodes contacting said at least one second section of saidthin-film photoresistor with the second surface of its contactingelectrode; each of said second plurality of microelectrodes beingconnected to one of said pad electrodes by said at least one secondsection of said thin-film resistor contacting said at least one padelectrode at the second side thereof.
 13. A microelectrode arrangementaccording to claim 12, including fiberoptics having at least one opticalfiber arranged in front of one of said microelectrodes for emittinglight onto said thin-film resistor for optically activating said one ofsaid microelectrodes.
 14. A microelectrode arrangement according toclaim 13, wherein said fiberoptics comprise a plurality of opticalfibers, each one of said microelectrodes having one of said opticalfibers associated therewith for optically activating said one of saidmicroelectrodes.
 15. A microelectrode arrangement according to claim 13,wherein each optical fiber is associated with a light source foremitting light into said optical fiber.